Source: Laboratory Guide to Proteins and Proteomics
Operation method
basic program
Principle
It is very convenient to use bacteria to obtain recombinant proteins for purification. Methods suitable for protein extraction from bacterial cells include ultrasonic treatment, glass bead milling, vanadium or quartz sand milling, high pressure shearing of cells in French pressurized tanks, and lysozyme treatment. These methods are suitable for the preparation of protein extracts from a variety of Gram-negative and Gram-positive bacteria. Enzymatic methods are usually used to disrupt the cells because the cells receive a relatively homogeneous treatment in suspension.
Materials and Instruments
Bacterial cells expressing target recombinant proteins Move ①Centrifuge at 4℃ and 3000g for 15min to collect bacteria. ② Wash the cells with lysis buffer to remove the residual culture medium. Repeat step ① and centrifuge to collect the washed cells. ③ Pour out the supernatant and weigh the wet weight of the precipitate. ④ Resuspend each gram of cell precipitate with about 3 ml of lysis buffer, and stir the suspension for 30 min at 4°C. If the precipitate is not completely suspended after 30 min, mix the suspension for 1 min at low speed with a Werin stirrer. ⑤ Add lysozyme to a final concentration of 1g/L and incubate for 35min at 4°C with gentle shaking. Increasing the concentration of lysozyme to 10 mg/ml accelerates cleavage. In this way, sufficient cleavage was obtained in 5 min at 4°C ⑥ Add the following reagents in sequence: NP-40 to a final concentration of 5g/L MgCl2 to a final concentration of 5 mmol/L DNaseI to a final concentration of 40/zg/ml The suspension was stirred for 30mm at 4°C to remove the sticky nucleic acids. The bacterial extract contained 40%~70% protein, 10%~30% nucleic acids, 2%~10% polysaccharides and 10%~15% lipids. The release of DNA during cell lysis often results in highly viscous extracts, which can cause serious trouble in the following chromatographic purification steps. In addition to treatment with DNaseI, positively charged compounds such as ichthyosin sulfate (to 5 mg/g precipitated wet weight) or polyethylenimine can also be added to remove DNA (accompanied by other nucleic acids and, in some cases, highly acidified proteins) from the cell extracts by neutralizing the solution. Lab tip: The method of removing DNA with positively charged compounds cannot be used for inclusion body extraction. This is because the precipitated DNA will be centrifuged off with the inclusion bodies. (vii) The suspension was centrifuged at 23,000 g for 30 min at 4°C. ⑧ Resuspend a small portion of the precipitate with Laemmli buffer containing DTT. ⑨ Analyze the soluble protein fraction (supernatant) and the precipitated fraction using analytical grade SDS-PAGE to find out where the target protein is present. If the target protein is located in the insoluble precipitation fraction, an inclusion body may be formed, and then the target protein will need to be solubilized and purified according to other protocols. If the target protein is in the supernatant, it is stored at 4°C for subsequent purification protocols. Caveat 1×Laemmli sample buffer:2% SDS10% glycerol60mmol/L Tris-Cl (pH 6.8)0.01% bromophenol blueIt is usually more convenient to make a 2× or 5× reservoir of Laemmli Sample Buffer. Add DTT to a final concentration of 100 mmol/L before use.Lysis buffer:50 mmol/L Tris-Cl (pH 7.4)250g/L sucroseMetal ions can be chelated by adding EDTA (to a final concentration of 10 mmol/L) to reduce metal ion protease activity. For more product details, please visit Aladdin Scientific website.
Dithiothreitol (DTT) DNaseI 1×Laemmli sample buffer Lysis buffer
Centrifuge